Equipment and method for feeding liquid gradient in nano/micro liquid chromatography

ABSTRACT

It is an object of the present invention to improve the performance of gradient liquid transfer in nano/micro liquid chromatographs when solvents are mixed. 
     In a gradient liquid transfer device and method for a nano/micro liquid chromatograph, an opening and closing unit ( 160 ) capable of opening and closing a solvent passage ( 150 ) for transferring a secondary solvent is provided in the vicinity of a solvent mixing section ( 152 ) in a liquid transfer system ( 130 ); a passage from a metering pump ( 146 ) of a liquid transfer section ( 142 ) to the opening and closing unit ( 160 ) is filled with the secondary solvent in advance; and an appropriate pressure is applied to the secondary solvent. Therefore, the entry of a primary solvent into the solvent passage for transferring the secondary solvent is suppressed. In another gradient liquid transfer device and method for a nano/micro liquid chromatograph, a liquid having a low compression rate is filled, in advance, in a part of a solvent passage subsequent to a metering pump of a liquid transfer section, and only the required amount of a secondary solvent is filled subsequent to the liquid. Therefore, the entry of the primary solvent into a solvent passage for transferring the secondary solvent is suppressed, and liquid-transfer performance is improved.

REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/JP03/09375 filed Jul. 24, 2003.

RELATED APPLICATIONS

The present application claims the benefit and priority of the JapanesePatent Application No. 2002-215415, filed in Japan on Jul. 24, 2002, thesubject matter of which is hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to gradient liquid transfer devices andmethods for nano/micro liquid chromatographs, and more-particularly, toan improvement of liquid transfer performance in solvent mixing.

2. Prior Art

Recently, high performance liquid chromatographs have served asrepresentative means for the separation and analysis of tracecomponents. FIG. 4 shows an example structure of such a chromatograph.In a high performance liquid chromatograph 10, a solvent is transferredby a pump 14 from a mobile-phase solvent tank 12 through a passage, andan injector 16 introduces a trace sample into the solvent in thepassage. The flow of the solvent leads the trace sample to a subsequentcolumn 18. The trace sample is separated into components at a controlledtemperature. The separated components are then detected by detectionmeans 20, such as an absorbance detector. A computer 22 processes andanalyzes A/D-converted detected signals, and also controls theconditions of the chromatograph.

Recently, a lower flow rate and a lower volume have been used toseparate infinitesimal components at high resolution. Nano/micro liquidchromatographs which u se a flow rate of several tens of micro litersper minute to a nano liter per minute have been developed.

FIG. 5 shows a conventional liquid transfer system used for gradientelution in such a nano/micro liquid chromatograph. In the liquidtransfer system 30, at a primary-solvent liquid transfer section 32, aprimary solvent is replenished from a primary-solvent tank 34 to ametering pump 36, and then, a valve 38 is switched to transfer theprimary solvent from the metering pump 36 through a primary-solventpassage 40 connected thereto.

At a secondary-solvent liquid transfer section 42, a secondary solventis replenished from a secondary-solvent tank 44 to a metering pump 46,and then a valve 48 is switched to transfer the secondary solvent fromthe metering pump 46 through a secondary-solvent passage 50 connectedthereto.

A three-way tee 52 (solvent mixing section) connected to theprimary-solvent passage 40 and to the secondary-solvent passage 50 mixesthe two solvents at a predetermined ratio. The mixed solvent istransferred to a subsequent separation system through a mixed-solventpassage 54. The mixing ratio of the two solvents is determined by theratio of the flow rates specified by the metering pumps, and iscontrolled by a control section such as a computer. With the mixingratio of the solvents being gradually changed, the mixed solvent istransferred to the subsequent separation system for gradient elution.

Due to the following two reasons, however, a considerable amount of theprimary solvent enters the secondary-solvent passage 50 when only theprimary solvent is transferred at a first stage.

First, since only a part (for example, from the pump 46 to the valve 48in FIG. 5) of the passage 50 is initially filled with the secondarysolvent, the primary solvent enters an empty part (for example, from thevalve 48 to the three-way tee 52 in FIG. 5) of the passage 50, which hasa considerable volume.

Secondly, the pressure of the liquid transfer system reaches, forexample, as high as several tens of kilograms per square centimetersbecause of the resistance of the separation system, which is subsequentto the liquid transfer system. The primary solvent having a highpressure and entering the secondary-solvent passage 50 pushes thesecondary solvent, which was previously stored at atmospheric pressure,to compress the secondary solvent. As a result, a further amount of theprimary solvent enters the secondary-solvent passage. When water is usedas the primary solvent and acetonitrile is used as the secondarysolvent, acetonitrile is compressed due to the pressure of the water,and a considerable amount of volume contraction occurs. Since nano/microliquid chromatographs have very small passage diameters and very smallpassage volumes, this solvent contraction also largely affects the entryof the primary solvent into the secondary-solvent passage.

When a considerable amount of the primary solvent enters thesecondary-solvent passage in this way, even if the operations of themetering pumps are controlled such that the transfer of the primarysolvent only is switched to the transfer of the mixed solvent at apredetermined time, actual switching is performed at a time later thanthe predetermined time, as shown in the graph of FIG. 6. In other words,it takes time for the pump 46 to push back the primary solvent which hasentered the secondary-solvent passage 50, the secondary solvent cannotbe mixed with the primary solvent during this time; and a time delaythus occurs. Therefore, the mixed solvent may be transferred to theseparation column with a delay, so that separation is performed late; oranalysis of the measurement results may be inaccurate, which is aproblem.

SUMMARY OF THE INVENTION

The present invention has been made in consideration of the foregoingissues of the conventional technology. It is an object of the presentinvention to improve the performance of gradient liquid transfer innano/micro liquid chromatographs when solvents are mixed.

The foregoing object is achieved in one aspect of the present inventionthrough the provision of a gradient liquid transfer device for anano/micro liquid chromatograph, provided with a primary-solvent liquidtransfer section having a metering pump for transferring a primarysolvent and a primary-solvent passage subsequent to the metering pump;at least one liquid transfer section having a metering pump fortransferring one other solvent and a solvent passage subsequent to themetering pump; a solvent mixing section connected to the passages and toa mixed-solvent passage for passing a mixed solvent made by mixing thesolvents supplied through the passages at a predetermined mixing ratioand for leading to a subsequent separation system; and a control sectionfor controlling the mixing ratio of the mixed solvent transferred to thesubsequent separation system, the gradient liquid transfer deviceincluding, in the vicinity of the solvent mixing section, opening andclosing means capable of opening and closing the solvent passage fortransferring the one other solvent.

In the gradient liquid transfer device for a nano/micro liquidchromatograph, a liquid connection section for connecting theprimary-solvent liquid transfer section and the at least one liquidtransfer section may be further included.

The foregoing object is achieved in another aspect of the presentinvention through the provision of a gradient liquid transfer device fora nano/micro liquid chromatograph, provided with a primary-solventliquid transfer section having a metering pump for transferring aprimary solvent and a primary-solvent passage subsequent to the meteringpump; at least one liquid transfer section having a metering pump fortransferring one other solvent and a solvent passage subsequent to themetering pump; a solvent mixing section connected to the passages and toa mixed-solvent passage for passing a mixed solvent made by mixing thesolvents supplied through the passages at a predetermined mixing ratioand for leading to a subsequent separation system; and a control sectionfor controlling the mixing ratio of the mixed solvent transferred to thesubsequent separation system, the gradient liquid transfer deviceincluding first storing means formed of the metering pump in the atleast one liquid transfer section and a part of the solvent passagesubsequent to the metering pump and filled in advance with a liquidhaving a low compression rate; and second storing means formed of a partof the solvent passage subsequent to the first storing means and filledwith a required amount of the one other solvent subsequent to the liquidin advance.

The foregoing object is achieved in still another aspect of the presentinvention through the provision of a gradient liquid transfer method fora nano/micro liquid chromatograph provided with a primary-solvent liquidtransfer section having a metering pump for transferring a primarysolvent and a primary-solvent passage subsequent to the metering pump;at least one liquid transfer section having a metering pump fortransferring one other solvent and a solvent passage subsequent to themetering pump; and a solvent mixing section connected to the passagesand to a mixed-solvent passage for passing a mixed solvent made bymixing the solvents supplied through the passages at a predeterminedmixing ratio and for leading to a subsequent separation system, thenano/micro liquid chromatograph gradually changing the mixing ratio ofthe mixed solvent and transferring the mixed solvent to the subsequentseparation system for gradient elution, and including, in the vicinityof the solvent mixing section, opening and closing means capable ofopening and closing the solvent passage for transferring the one othersolvent, and the gradient liquid transfer method including a first stepof closing the opening and closing means, of filling, in advance, apassage from the metering pump of the at least one liquid transfersection to the opening and closing means with the one other solvent, andof applying an appropriate pressure to the one other solvent; a secondstep of transferring the primary solvent to the subsequent separationsystem from the primary-solvent liquid transfer section through thesolvent mixing section; and a third step of opening the opening andclosing means, of transferring the one other solvent to the solventmixing section at a predetermined flow rate, and of transferring themixed solvent having the predetermined mixing ratio of the primarysolvent and the one other solvent to the subsequent separation system.

The gradient liquid transfer method may be configured such that thenano/micro liquid chromatograph further includes a liquid connectionsection for connecting the primary-solvent liquid transfer section andthe at least one liquid transfer section, and pressure generated in theprimary-solvent liquid transfer section in the second step is applied tothe at least one liquid transfer section to apply the appropriatepressure to the one other solvent in the first step.

The foregoing object is achieved in yet another aspect of the presentinvention through the provision of a gradient liquid transfer method fora nano/micro liquid chromatograph provided with a primary-solvent liquidtransfer section having a metering pump for transferring a primarysolvent and a primary-solvent passage subsequent to the metering pump;at least one liquid transfer section having a metering pump fortransferring one other solvent and a solvent passage subsequent to themetering pump; and a solvent mixing section connected to the passagesand to a mixed-solvent passage for passing a mixed solvent made bymixing the solvents supplied through the passages at a predeterminedmixing ratio and for leading to a subsequent separation system, thenano/micro liquid chromatograph gradually changing the mixing ratio ofthe mixed solvent and transferring the mixed solvent to the subsequentseparation system for gradient elution, and the gradient liquid transfermethod including a first step of filling, in advance, a liquid having alow compression rate in a part of the solvent passage subsequent to themetering pump of the at least one liquid transfer section; a second stepof filling a required amount of the one other solvent, subsequent to theliquid, in the solvent passage; a third step of transferring the primarysolvent to the subsequent separation system from the primary-solventliquid transfer section through the solvent mixing section; and a fourthstep of transferring the one other solvent to the solvent mixing sectionat a predetermined flow rate, and of transferring the mixed solventhaving the predetermined mixing ratio of the primary solvent and the oneother solvent to the subsequent separation system.

In any of the above-described gradient liquid transfer devices andmethods, it is preferred that the metering pumps be syringe-typemetering pumps, each transferring the entire solvent by pushing thesyringe in a single stroke.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view showing a liquid transfer system which employs a methodaccording to a first embodiment of the present invention.

FIG. 2 is a view showing a liquid transfer system which employs a methodaccording to a second embodiment of the present invention.

FIG. 3 is a view showing a syringe pump used in a method according tothe present invention.

FIG. 4 is a view showing a high performance liquid chromatograph.

FIG. 5 is a view showing a conventional liquid transfer system.

FIG. 6 is a view showing a switching time when the transfer of a primarysolvent only is switched to the transfer of a mixed solvent.

FIG. 7 is a view showing a pressure gage.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Embodiments of the present invention will be described below byreferring to the drawings.

First Embodiment

FIG. 1 shows an outline structure of a liquid transfer system whichemploys a method according to a first embodiment of the presentinvention. Elements corresponding to those in the conventionaltechnology have the same reference numerals as those assigned to theelements in the conventional technology plus 100. In the liquid transfersystem 130, at a primary-solvent liquid transfer section 132, a primarysolvent is replenished from a primary-solvent tank 134 to a meteringpump 136, and then, a valve 138 is switched to transfer the primarysolvent from the metering pump 136 through a primary-solvent passage 140connected thereto.

At a secondary-solvent liquid transfer section 142, a secondary solventis replenished from a secondary-solvent tank 144 to a metering pump 146,and then, a valve 148 is switched to transfer the secondary solvent fromthe metering pump 146 through a secondary-solvent passage 150 connectedthereto.

In the present embodiment, capillaries having a passage diameter of 250μm or smaller are used for the primary-solvent passage 140 and thesecondary-solvent passage 150.

A three-way tee (solvent mixing section) 152 connected to theprimary-solvent passage 140 and to the secondary-solvent passage 150mixes the two solvents at a predetermined ratio. The mixed solvent istransferred to a subsequent separation system through a mixed-solventpassage 154. The mixing ratio of the two solvents is determined by theratio of the flow rates specified by the metering pumps, and iscontrolled by a control section such as a computer. With the mixingratio of the solvents being gradually changed, the mixed solvent istransferred to the subsequent separation system for gradient elution.

The present embodiment is characterized by providing, in the vicinity ofthe solvent mixing section 152, opening and closing means capable ofopening and closing the solvent passage 150 which transfers the secondsolvent. In the present embodiment, a valve 160 is used as the openingand closing means. Since the opening and closing means is provided inthe vicinity of the solvent mixing section 152, the entry of the primarysolvent into the secondary-solvent passage 150, which is the problemdescribed above, can be greatly suppressed. More specifically, theprimary solvent can enter the secondary-solvent passage 150 just as faras the closest end of the valve 160, which is a small volume, and cannotenter the other part of the passage between the valve 160 and themetering pump 146, which occupies a relatively large volume. With theuse of the opening and closing means, the method according to thepresent invention has the following steps.

First Step

The valve 160 is closed to block the passage 150, and the passagebetween the metering pump 146 and the valve 160 is filled with thesecondary solvent. An appropriate pressure is given to the secondarysolvent by driving the metering pump 146. With the pressure beingapplied in advance, as in this case, the volume contraction of thesecondary solvent caused by the pressure of the primary solvent appliedwhen the valve 160 is opened can be suppressed. Therefore, the amount ofthe primary solvent entering the passage 150 is reduced accordingly. Thepressure being applied in advance to the secondary solvent isappropriately determined according to conditions such as the solventused.

Second Step

The primary solvent is transferred from the primary-solvent liquidtransfer section 132 to the subsequent separation system through thesolvent mixing section 152. The pressure of the liquid transfer systemgradually increases due to the resistance of the subsequent separationsystem. In the present embodiment, in order to quickly reach a highpressure (20 kg/cm², for example,), the primary solvent is transferredfirst at a rate of 500 μl/min, and later, when the high pressure isobtained, at a rate of 500 nl/min.

Third Step

The valve 160 is opened to transfer the secondary solvent to the solventmixing section 152 at a predetermined flow rate. The mixed solventhaving a predetermined mixing ratio of the primary solvent and thesecondary solvent is transferred to the subsequent separation system. Inthe, present embodiment, the primary solvent entering the passage 150 islimited to a small volume as far as the valve 160, and in addition,since the pressure is given in advance to the secondary solvent, theentry of the primary solvent into the passage 150 caused by thecompression of the secondary solvent is also suppressed. Therefore, adelay from the predetermined time when the transfer of the primarysolvent only should be switched to the transfer of the mixed solvent canbe reduced.

The present embodiment is further characterized by providing a liquidconnection section 170 for connecting the primary-solvent liquidtransfer section 132 and the secondary-solvent liquid transfer section142. In the first step described above, it is necessary to drive thepump 146 to apply the pressure to the secondary solvent. When the liquidconnection section 170 is provided, the pressure of the primary-solventliquid transfer section 132 obtained after the transfer of the primarysolvent is started is conveyed to the secondary-solvent liquid transfersection 142. Since the same pressure is applied to the secondary solventfilling the section from the pump 146 to the valve 160, there is no needto perform any special operation to obtain a pressure balance.

In the present embodiment, the liquid connection section 170 is providedwith capillaries 172 and 174 for storing the two solvents, a valve 176,and pressure gages 178 and 180. The primary solvent is stored in advancein the capillary 172, and the secondary solvent is stored in advance inthe capillary 174. Then, the primary solvent is transferred to thesubsequent separation system from the primary-solvent liquid transfersection 132 through the solvent mixing section 152. The pressure of theprimary-solvent liquid transfer section 132 is gradually increased untilit reaches a high pressure (20 kg/cm², for example). When the valve 176is opened, since the primary solvent and the secondary solvent makecontact in the liquid connection section 170, the high pressure isconveyed to the second-solvent liquid transfer section 142, and the samepressure is applied to the secondary solvent filling the section up tothe closed valve 160. Therefore, the same effect as in the first stepdescribed above is obtained.

Since the capillaries 172 and 174 have sufficient volumes, the boundarybetween the primary solvent and the secondary solvent does not reach thepump 146 or the passage connected thereto. In addition, these solventsare not mixed. Because the valve 160 is opened at the start of the thirdstep, the valve 176 may be closed to block the passage, if necessary. Itis also preferred that a drain 184 be connected to the valve 176 througha stop valve 182.

When three or more types of solvents are used, a liquid transfersection(s) for transferring one other solvent(s) can be connected to thesolvent mixing section 152 in addition to the liquid transfer section142. In this case, any two of a plurality of liquid transfer sectionscan be connected.

Pressure Gages

FIG. 7 shows an outline structure of the pressure gages 178 and 180 usedin FIG. 1. In FIG. 7, when a mobile-phase solvent is transferred at aconstant flow rate, the resistance of a wall surface 406 against thesolvent flow occurs at a coupling section between a solvent passage 402and a capillary 404. With this resistance, the pressure against thepassage wall surfaces is increased around the coupling section due tothe transfer of pressurized solvent in a flow direction 408. Theincreased pressure against the wall surfaces is detected by a pressuregage 410.

Since nano/micro liquid chromatographs use very small amounts ofsolvents, it is difficult to check whether the solvents are transferredcorrectly during measurement, more specifically whether the solvents arenot leaked, or whether an extraordinarily high pressure due to cloggingdoes not occur. Therefore, such a pressure gage is provided at thepassage to monitor the liquid pressure to check whether the solvent istransferred correctly. More specifically, when the solvent istransferred correctly at a constant flow rate, a constant pressure isdetected. If the solvent is leaked, such leakage is indicated by apressure reduction. If an extraordinarily high pressure occurs due toclogging, it can be detected by the pressure gage.

Second Embodiment

FIG. 2 shows an outline structure of a liquid transfer system whichemploys a method according to a second embodiment of the presentinvention. Elements corresponding to those in the first embodiment havethe same reference numerals as those assigned to the elements in thefirst embodiment plus 100, and a description thereof is omitted.

The present embodiment is characterized in that a liquid having a lowcontraction rate with respect to pressure is filled, in advance, in apart of a solvent passage 250 subsequent to a metering pump 246 in asecond-solvent liquid transfer section 242. With this liquid, the effectof the volume contraction of a secondary solvent caused by pressure isconsiderably suppressed. The liquid transfer method according to thepresent embodiment has the following steps.

First Step

The liquid having the low compression rate is filled, in advance, in thepart of the solvent passage 250 subsequent to the metering pump 246. InFIG. 2, for example, a passage (first storing means) from the meteringpump 246 to a six-way valve 294 is filled with such a liquid by usingthe metering pump 246 and a tank 244 which stores the liquid.

Second Step

The secondary solvent is transferred from a secondary-solvent tank 290by a pump 292 to be filled, in advance, in a passage 296 (second storingmeans) between port “a” and port “b” of the six-way valve 294. Thepassage 296 needs to have a volume corresponding to the amount of thesecondary solvent required for measurement. The six-way valve 294 isswitched to paths indicated by dotted lines in FIG. 2 to fill thepassage 250 with the secondary solvent following the liquid having thelow compression rate.

Third Step

A primary solvent is transferred from a primary-solvent liquid transfersection 232 to a subsequent separation system through a solvent mixingsection 252. The pressure of the liquid transfer system graduallyincreases due to the resistance of the subsequent separation system. Theprimary solvent entering the passage 250 compresses the secondarysolvent filled there in advance. However, since the passage from thepump 246 to the six-way valve 294 is filled with the liquid having thelow compression rate, volume contraction can be suppressed in thispassage having a relatively large volume. In other words, whereas, inthe conventional technology shown in FIG. 5, the entire passage from thepump is filled with the secondary solvent and the secondary solvent issubjected to volume contraction due to pressure, in the presentembodiment shown in FIG. 2, although the amount of the secondary solventrequired for measurement is compressed, compression is only applied tothat amount, and the entry of the primary solvent into the passage 250caused by the volume contraction is considerably suppressed.

It is preferred that, as the liquid having the low compression rate, aliquid having a lower compression rate than the secondary solvent beappropriately selected according to other measurement conditions.

Fourth Step

The secondary solvent is transferred to the solvent mixing section 252at a predetermined flow rate. The mixed solvent having a predeterminedmixing ratio of the primary solvent and the secondary solvent istransferred to the subsequent separation system. As described above,since only the actual amount of the secondary solvent required formeasurement is used in the present embodiment, a delay from apredetermined time when the transfer of the primary solvent only shouldbe switched to the transfer of the mixed solvent can be reduced.

When three or more types of mobile-phase solvents are used, a liquidtransfer section(s) for transferring one other solvent(s) can beconnected to the solvent mixing section 252 in addition to the liquidtransfer section 242.

Metering Pump

A syringe pump preferably used as a metering pump in the presentinvention will be described below by referring to FIG. 3. In a syringepump 300 shown in FIG. 3, a solvent introduced in advance into a head304 is discharged to a passage 306 by driving a syringe 302. Sincenano/micro liquid chromatographs use a trace amount of a solvent forseparation, the entire solvent is transferred by pushing the syringe ina single stroke with the use of the rotation of a screw 310 driven by amotor 308. Therefore, the transfer is performed without generating apulsating flow caused by the repetitions of discharges and suctions.

When such a syringe pump is used, a temperature change of the pump maycause a change in the flow rate. Therefore, it is preferred that meansfor maintaining a constant temperature of the pump be used.

Even when appropriate temperature adjustment means is used, however, achange in the flow rate caused by the temperature change of the pumpcannot be ignored in some cases. In our examination, it is found that,in order to suppress a change in the flow rate, it is effective toreduce the volume of the syringe introduced into the head 304 alwayswhile it is transferring liquid.

It is thought that the reason why the temperature change of the pumpcauses a change in the flow rate is a variation in the solvent volumecaused by the temperature change of the pump, or a variation in thesyringe volume, or both. The density of a solvent is increased by about0.1% as the temperature increases by 1° C. The volume of the syringe mayalso increase with temperature. Therefore, reducing the volume of thesyringe should reduce those variations. It is especially preferred thatthe volume be set to 1000 μl or smaller.

As described above, according to a gradient liquid transfer device andmethod of the present invention, the following advantages are obtained.

(1) The opening and closing means capable of opening and closing thesecondary-solvent passage is provided in the vicinity of the solventmixing section; the passage from the metering pump of thesecondary-solvent liquid transfer section to the opening and closingmeans is filled with the secondary solvent in advance; and anappropriate pressure is applied to the secondary solvent. Therefore, theentry of the primary solvent into the secondary-solvent passage issuppressed. In this case, when the liquid connection section forconnecting the primary-solvent liquid transfer section and thesecondary-solvent liquid transfer section is provided, since thepressure of the primary solvent is applied to the secondary solvent, anoperation for applying an appropriate pressure to the secondary solventcan be omitted.

(2) The liquid having the low compression rate is filled, in advance, ina part of the solvent passage subsequent to the metering pump of theliquid transfer section, and only the required amount of the secondarysolvent is filled subsequent to the liquid. Therefore, the entry of theprimary solvent into the secondary-solvent passage is suppressed.

1. A gradient liquid transfer method for a nano/micro liquidchromatograph provided with: a primary-solvent liquid transfer sectionhaving a metering pump for transferring a primary solvent and aprimary-solvent passage subsequent to the metering pump; at least oneliquid transfer section having a metering pump for transferring oneother solvent and a solvent passage subsequent to the metering pump; anda solvent mixing section connected to the passages and to amixed-solvent passage for passing a mixed solvent made by mixing thesolvents supplied through the passages at a predetermined mixing ratioand for leading to a subsequent separation system, the nano/micro liquidchromatograph gradually changing the mixing ratio of the mixed solventand transferring the mixed solvent to the subsequent separation systemfor gradient elution, and comprising, in the vicinity of the solventmixing section, opening and closing means capable of opening and closingthe solvent passage for transferring the one other solvent, and thegradient liquid transfer method comprising: a first step of closing theopening and closing means, of filling, in advance, a passage from themetering pump of the at least one liquid transfer section to the openingand closing means with the one other solvent, and of applying anappropriate pressure to the one other solvent; a second step oftransferring the primary solvent to the subsequent separation systemfrom the primary-solvent liquid transfer section through the solventmixing section; and a third step of opening the opening and closingmeans, of transferring the one other solvent to the solvent mixingsection at a predetermined flow rate, and of transferring the mixedsolvent having the predetermined mixing ratio of the primary solvent andthe one other solvent to the subsequent separation system.
 2. A gradientliquid transfer method for a nano/micro liquid chromatograph accordingto claim 1, the nano/micro liquid chromatograph further comprising aliquid connection section for connecting the primary-solvent liquidtransfer section and the at least one liquid transfer section, whereinpressure generated in the primary-solvent liquid transfer section in thesecond step is applied to the at least one liquid transfer section toapply the appropriate pressure to the one other solvent in the firststep.
 3. A gradient liquid transfer method for a nano/micro liquidchromatograph according to claim 1 wherein the metering pumps aresyringe-type metering pumps, each transferring the entire solvent bypushing the syringe in a single stroke.